Novel Truncating Variant c.1222DupC in RBM20 Causes Cardiomyopathy Consistent With Haploinsufficiency

RBM20 (RNA binding motif protein 20) is a cardiac splicing factor responsible for the splicing of several cardiac genes such as titin (TTN), triadin (TRDN), ryanodine receptor 2 (RYR2), PDZ and LIM domain protein 1 (PDLIM1), and calcium/calmodulin-dependent protein kinase II (CAMK2D). Pathogenic variants in RBM20 are a major cause of familial dilated cardiomyopathy, and lead to missplicing of RBM20 target genes.

We identified a patient with a novel RBM20 variant, and expressed the human and mouse-equivalent variant in neonatal rat cardiomyocytes and HEK293 cells. We performed splicing assays, and assessed protein expression and stability. Furthermore, we generated heterozygous RBM20-c.1222DupC human induced pluripotent stem cells, differentiated these into human induced pluripotent stem cell-derived cardiomyocytes, and evaluated splicing changes and calcium handling.

We describe a novel heterozygous truncating variant, RBM20-c.1222DupC, identified in a patient with mitral valve prolapse and late-onset familial dilated cardiomyopathy. The variant introduces a premature termination codon and generates a truncated protein of ≈55 kDa in vitro. Splicing assays demonstrated complete loss of activity and no dominant-negative effect on wild-type RBM20. The truncated protein localized to both the cytoplasm and nucleus, partially colocalizing with wild-type RBM20, despite lacking the RS and RRM domains. Western blot analysis of endogenous RBM20 in human induced pluripotent stem cell-derived cardiomyocytes carrying the variant revealed a strong reduction in RBM20 protein levels. Reverse transcriptase-polymerase chain reaction revealed splicing defects in canonical RBM20 targets, and RNA sequencing identified widespread splicing abnormalities, including in established RBM20 targets (TTN, RYR2, CAMK2D, and CACNA1G). Finally, we observed increased calcium transients.

Together, these findings establish RBM20 c.1222DupC as a truncating variant that causes dilated cardiomyopathy likely through haploinsufficiency.